Cell transfecting formulations of small interfering RNA related compositions and methods of making and use

ABSTRACT

Compositions incorporating small interfering ribonucleic acid (siRNA) and certain lipid-conjugated polyamide compound-based delivery vehicles that are particularly useful in the delivery siRNA and other polynucleotides to cells. Also, methods of making and using the compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/530,953, filed Dec. 19, 2003, which is hereby incorporated herein inits entirety.

FIELD OF THE INVENTION

This invention relates to compositions incorporating small interferingribonucleic acid (siRNA) with lipid-conjugated polyamide compounds,methods for making them, as well as methods for their use in thedelivery of siRNA to cells. The invention also relates to a novel classof lipid-conjugated polyamide compounds suitable for use in the deliveryof polynucleotides, including siRNA, to cells

BACKGROUND OF THE INVENTION

RNA interference refers to the phenomenon of the presence of doublestranded RNA in a cell eliminating the expression of a gene having thesame sequence, while leaving the expression of other unrelated genesundisturbed. This phenomenon, also known as “post transcriptional genesilencing” or “RNA silencing” has been noted in plants for some time,but has only more recently been recognized in animals. Fire et al.,Nature, 391, 806 (1998). The discovery of this functionality suggeststhe possibility of powerful research tools for stopping the productionof a specific protein and gene-specific therapeutics operating by thismechanism.

Details of the RNA interference (RNAi) mechanism have recently beenelucidated. The presence of long dsRNAs in cells stimulates the activityof a ribonuclease III enzyme known to as Dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as smallinterfering RNAs (siRNA) (Berstein et al., Nature, 409, 363 (2001)).Small interfering RNAs derived from Dicer activity are typically about21-23 nucleotides in length. The RNAi response also features anendonuclease complex containing an siRNA, commonly referred to as anRNA-induced silencing complex (RISC). The RISC complex mediates cleavageof single stranded RNA having sequence complementary to the antisensestrand of the siRNA duplex. Cleavage of the target RNA takes place inthe middle of the region complementary to the antisense strand of thesiRNA duplex. Elbashir et al., Genes Dev., 15, 188 (2001).

One potential impediment to harnessing the RNAi phenomenon in mammaliancells is that the presence of long dsRNAs in these cells also stimulatesan interferon response that results in non-specific cleavage of mRNA bya ribonuclease. However, it has been shown that chemically synthesized21-meric small interfering RNAs (siRNAs) effectively suppress geneexpression in several human cell lines without eliciting an interferonresponse. Elbashir et al., Nature, 411, 494 (2001). In particular,synthetic siRNAs have been found to be most active when containing 21nucleotide duplexes with two TT nucleotide 3′-overhangs. Elbashir etal., EMBO J., 20, 6877 (2001).

SiRNA's characteristics of high specificity, resistance toribonucleases, non-immunogenicity and potency suggest tremendouspotential as a cell transfection agent for research and therapeuticapplications. A variety of strategies exist for delivery of nucleic acidcompositions to cells. However, technical difficulties have beenencountered in transfecting siRNA into cells. Viral vectors providerelatively efficient delivery, but in some cases present safety problemsdue to the risk of immunological complications or unwanted propagationin the subject. Adenoviral vectors have shown certain advantages in thatthey do not integrate into the genome of the cell and can be transducedinto resting cells. However, all of these vectors must be prepared bytime-consuming recombinant DNA techniques. Oligonucleotides may also bedelivered to cells via chemical transfection agents, which have been thesubject of much recent work. These agents include polycationicmolecules, such as polylysine, and cationic lipids. The liposomalcomposition Lipofectin® (Felgner et al., PNAS 84:7413, 1987), containingthe cationic lipid DOTMA(N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) and theneutral phospholipid DOPE (dioleyl phosphatidyl ethanolamine), is widelyused. Other methods, such as calcium phosphate mediated transfection,can be used to deliver the oligonucleotides to cells according toreported procedures. However, there is a need for effective, nontoxicsiRNA transfection agents that are easy to use and applicable to manycell types.

SUMMARY OF THE INVENTION

To achieve the foregoing, the present invention provides compositionsincorporating small interfering ribonucleic acid (siRNA) and certainlipid-conjugated polyamide compound-based delivery vehicles that areparticularly useful in the delivery of polynucleotides, including siRNA,to cells.

In one aspect, the invention provides compositions incorporating smallinterfering ribonucleic acid (siRNA) and lipid-conjugated polyamidecompounds having the general formula:R_(a)—[(NR₁—W—CO)_(n)]_(m)—R_(c)  (I)

-   -   wherein n is an integer selected from 1 to about 48 and m is an        integer selected from about 2 to about 48,    -   wherein R₁ for each monomeric unit, —(NR₁—W—CO)—, and R_(a) are        independently selected from the group consisting of a hydrogen        atom; a hydroxy group; an amino group; a carboxyl group; a        sulfonyl group; —SH; an optionally substituted, branched or        straight chain aliphatic group having 1 to 8 carbon atoms in a        backbone structure that optionally contains nitrogen, oxygen,        sulfur, and phosphorus, wherein said aliphatic group optionally        has one or more double or triple bonds; an optionally        substituted aryl group having 3 to 12 carbon atoms in a backbone        structure that optionally contains nitrogen, oxygen, sulfur, and        phosphorus; an optionally substituted arylalkyl group having 3        to 12 carbon atoms in a backbone structure that optionally        contains nitrogen, oxygen, sulfur, and phosphorus, wherein the        alkyl group of said arylalkyl optionally has one or more double        or triple bonds; and a lipid moiety that is optionally bonded to        linker moiety,    -   wherein R₁ is not a hydrogen atom for at least one monomeric        unit,    -   wherein R_(c) is selected from a hydrogen atom; a hydroxy group;        an amino group; a hydrazine group; a sulfonyl group; —SH; an        optionally substituted, branched or straight chain aliphatic        group having 1 to 8 carbon atoms in a backbone structure that        optionally contains nitrogen, oxygen, sulfur, and phosphorus,        wherein said aliphatic group optionally has one or more double        or triple bonds; an optionally substituted aryl group having 3        to 12 carbon atoms in a backbone structure that optionally        contains nitrogen, oxygen, sulfur, and phosphorus; an optionally        substituted arylalkyl group having 3 to 12 carbon atoms in a        backbone structure that optionally contains nitrogen, oxygen,        sulfur, and phosphorus, wherein the alkyl group of said        arylalkyl optionally has one or more double or triple bonds; and        a lipid moiety that is optionally bonded to a linker moiety,    -   wherein when R₁, R_(a), or R_(c) is an aryl or arylalkyl group        having fewer than 5 carbon atoms in a backbone structure, said        backbone structure further comprises one or more oxygen and/or        nitrogen atoms,    -   wherein W for each monomeric unit is independently selected from        an optionally substituted, branched or straight chain divalent        moiety having from 1 to about 50 atoms and optionally, one or        more double or triple bonds in a backbone that contains carbon        and optionally contains nitrogen, oxygen, sulfur, and        phosphorus, wherein said optional substitution of W may be a        lipid moiety that is optionally bonded to a linker moiety,    -   wherein said lipid moiety is a hydrophobic or amphipathic moiety        selected from the group consisting of:    -   (i) optionally substituted aryl or arylalkyl moieties having        from about 14 to about 50 carbon atoms in a backbone structure        that optionally contains nitrogen, oxygen, sulfur, and        phosphorus, wherein the alkyl group of said arylalkyl optionally        has one or more double or triple bonds; and    -   (ii) optionally substituted, branched or straight chain        aliphatic moieties having from about 10 to about 50 carbon atoms        in a backbone structure that optionally contains nitrogen,        oxygen, sulfur, and phosphorus, wherein said aliphatic moieties        optionally have one or more double or triple bonds, and    -   wherein at least one of R_(a), R_(c), W for a single monomeric        unit and R₁ for a single monomeric unit comprises a lipid        moiety.

In a particular embodiment, the invention provides a compositionincluding a siRNA in a pharmaceutically acceptable vehicle. Thecomposition may be useful for delivering the siRNA to a cell, in vitroor in vivo, to inhibit expression of a gene of interest. The vehicleincludes one or more lipid-cationic peptoid conjugates of the formula:L-X—[N(CH₂CH₂NH₂)CH₂(C═O)—N(CH₂CH₂R)CH₂(C═O)N(CH₂CH₂R)CH₂(C═O)]₃—NH₂  (VI)and positional isomers where

-   -   L is selected from a non-steroidal lipid moiety comprising at        least one fatty alkyl or alkenyl chain between about 8 and 24        carbon atoms in length, and a sterol moiety;    -   each group R is independently selected from alkyl, aminoalkyl,        and aralkyl, and    -   X is selected from the group consisting of a direct bond, an        oligopeptide, a substantially linear alkyl chain from 2 to about        30 bonds in length, and a substantially linear chain from 2 to        about 30 bonds in length consisting of alkyl bonds and one or        more linkages selected from the group consisting of ester,        amide, carbonate, carbamate, disulfide, peptide, and ether.

When L is a non-sterol lipid moiety (that is, a lipid moiety that is notor does not contain a sterol group, such as a phospholipid group (i.e.,ROOCCH₂CH(COOR)CH₂OP(O)₂O—), the lipid-cationic polyamide conjugate isreferred to herein as a “lipitoid.” When L is a sterol moiety, (that is,a lipid moiety that is or contains a sterol group, such as a cholesterolgroup), the lipid-cationic polyamide conjugate is referred to herein asa “cholesteroid.” The lipid-cationic peptoid conjugate in a compositionof the present invention may be a lipitoid, a cholesteroid, or, in oneimportant embodiment, a combination thereof.

In specific embodiments, R is isopropyl or 4-methoxyphenyl. A singlelipitoid or cholesteroid may include different groups R, or they may bethe same within the molecule.

The compositions of the invention result in efficient delivery of thebiologically active siRNA to mammalian cells effective to knockout themRNA of a target gene.

In another aspect, a method of inhibiting expression of a target gene ina subject, which involves administering to the subject a composition asdescribed above, in which one strand of the siRNA duplex has anucleotide sequence comprised in a mRNA derived from the target gene isprovided. In a specific embodiment, a strand of the siRNA duplexincludes a sequence represented by SEQ ID NO: 1, disclosed herein, andthe target gene/mRNA is Akt1.

In another aspect, the invention provides a polynucleotide deliveryvehicle composed of a mixture of at least one lipitoid and onecholesteroid. This combination lipitoid/cholesteroid delivery vehicle issuitable for delivery of a variety of polynucleotides such as plasmidDNA, antisense oligonucleotides and siRNA, to cells in compositionsincorporating such polynucleotides and the combinationlipitoid/cholesteroid delivery vehicle.

Methods of manufacturing compounds and compositions described herein areprovided and contemplated to fall within the scope of the invention asis the use of the compositions in methods for manufacturing medicamentsfor use in the methods of the invention.

These and other objects and features of the invention will become morefully apparent when the following detailed description of the inventionis read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a selection of lipid-cationic peptoid conjugates(“lipitoids” and “cholesteroids”) useful as siRNA carriers incompositions and methods of the invention.

FIG. 2 is a plot showing loss of Akt1 expression when siRNA directedagainst Akt1 mRNA is transfected into MDA435 breast cancer cells usingtransfection compositions in accordance with the present invention.

FIG. 3 is a plot showing luciferase activity in cells treated withtransfection mixtures which were prepared using siRNA against fireflyluciferase and several different delivery vehicles in accordance withthe present invention.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The materials and associated techniques and apparatuses of the presentinvention will now be described with reference to several embodiments.Important properties and characteristics of the described embodimentsare illustrated in the structures in the text. While the invention willbe described in conjunction with these embodiments, it should beunderstood that the invention it is not intended to be limited to theseembodiments. On the contrary, it is intended to cover alternatives,modifications, and equivalents as may be included within the spirit andscope of the invention as defined by the appended claims. In thefollowing description, numerous specific details are set forth in orderto provide a thorough understanding of the present invention. Thepresent invention may be practiced without some or all of these specificdetails. In other instances, well known process operations have not beendescribed in detail in order not to unnecessarily obscure the presentinvention.

Introduction

The present invention provides compositions incorporating smallinterfering ribonucleic acid (siRNA) and lipid-conjugatedpolyamide-based delivery vehicles that are particularly useful in thedelivery of siRNA to cells. In another aspect, the invention provides apolynucleotide delivery vehicle composed of a mixture of at least onelipitoid and one cholesteroid. The delivery vehicle is suitable fordelivery of a variety of polynucleotides, such as plasmid DNA, antisenseoligonucleotides and siRNA, to cells.

In one aspect, the invention provides compositions incorporating smallinterfering ribonucleic acid (siRNA) and lipid-conjugated polyamidecompounds having the general formula:Ra—[(NR₁—W—CO)_(n)]_(m)—R_(c)  (I)

-   -   wherein n is an integer selected from 1 to about 48 and m is an        integer selected from about 2 to about 48,    -   wherein R₁ for each monomeric unit, —(NR₁—W—CO)—, and R_(a) are        independently selected from the group consisting of a hydrogen        atom; a hydroxy group; an amino group; a carboxyl group; a        sulfonyl group; —SH; an optionally substituted, branched or        straight chain aliphatic group having 1 to 8 carbon atoms in a        backbone structure that optionally contains nitrogen, oxygen,        sulfur, and phosphorus, wherein said aliphatic group optionally        has one or more double or triple bonds; an optionally        substituted aryl group having 3 to 12 carbon atoms in a backbone        structure that optionally contains nitrogen, oxygen, sulfur, and        phosphorus; an optionally substituted arylalkyl group having 3        to 12 carbon atoms in a backbone structure that optionally        contains nitrogen, oxygen, sulfur, and phosphorus, wherein the        alkyl group of said arylalkyl optionally has one or more double        or triple bonds; and a lipid moiety that is optionally bonded to        linker moiety,    -   wherein R₁ is not a hydrogen atom for at least one monomeric        unit,    -   wherein R_(c) is selected from a hydrogen atom; a hydroxy group;        an amino group; a hydrazine group; a sulfonyl group; —SH; an        optionally substituted, branched or straight chain aliphatic        group having 1 to 8 carbon atoms in a backbone structure that        optionally contains nitrogen, oxygen, sulfur, and phosphorus,        wherein said aliphatic group optionally has one or more double        or triple bonds; an optionally substituted aryl group having 3        to 12 carbon atoms in a backbone structure that optionally        contains nitrogen, oxygen, sulfur, and phosphorus; an optionally        substituted arylalkyl group having 3 to 12 carbon atoms in a        backbone structure that optionally contains nitrogen, oxygen,        sulfur, and phosphorus, wherein the alkyl group of said        arylalkyl optionally has one or more double or triple bonds; and        a lipid moiety that is optionally bonded to a linker moiety,    -   wherein when R₁, R_(a), or R_(c) is an aryl or arylalkyl group        having fewer than 5 carbon atoms in a backbone structure, said        backbone structure further comprises one or more oxygen and/or        nitrogen atoms,    -   wherein W for each monomeric unit is independently selected from        an optionally substituted, branched or straight chain divalent        moiety having from 1 to about 50 atoms and optionally, one or        more double or triple bonds in a backbone that contains carbon        and optionally contains nitrogen, oxygen, sulfur, and        phosphorus, wherein said optional substitution of W may be a        lipid moiety that is optionally bonded to a linker moiety,    -   wherein said lipid moiety is a hydrophobic or amphipathic moiety        selected from the group consisting of:    -   (i) optionally substituted aryl or arylalkyl moieties having        from about 14 to about 50 carbon atoms in a backbone structure        that optionally contains nitrogen, oxygen, sulfur, and        phosphorus, wherein the alkyl group of said arylalkyl optionally        has one or more double or triple bonds; and    -   (ii) optionally substituted, branched or straight chain        aliphatic moieties having from about 10 to about 50 carbon atoms        in a backbone structure that optionally contains nitrogen,        oxygen, sulfur, and phosphorus, wherein said aliphatic moieties        optionally have one or more double or triple bonds, and    -   wherein at least one of R_(a), R_(c), W for a single monomeric        unit and R₁ for a single monomeric unit comprises a lipid        moiety.

In a particular embodiment, the invention provides a compositionincluding a siRNA in a pharmaceutically acceptable vehicle. Thecomposition may be useful for delivering the siRNA to a cell, in vitroor in vivo, to inhibit expression of a gene of interest. The vehicleincludes one or more lipid-cationic peptoid conjugates of the formula:L-X—[N(CH₂CH₂NH₂)CH₂(C═O)—N(CH₂CH₂R)CH₂(C═O)—N(CH₂CH₂R)CH₂(C═O)]₃—NH₂  (VI)and positional isomers where

-   -   L is selected from a non-steroidal lipid moiety comprising at        least one fatty alkyl or alkenyl chain between about 8 and 24        carbon atoms in length, and a sterol moiety;    -   each group R is independently selected from alkyl, aminoalkyl,        and aralkyl, and    -   X is selected from the group consisting of a direct bond, an        oligopeptide, a substantially linear alkyl chain from 2 to about        30 bonds in length, and a substantially linear chain from 2 to        about 30 bonds in length consisting of alkyl bonds and one or        more linkages selected from the group consisting of ester,        amide, carbonate, carbamate, disulfide, peptide, and ether.

When L is a non-sterol lipid moiety (that is, a lipid moiety that is notor does not contain a sterol group, such as a phospholipid group (i.e.,ROOCCH₂CH(COOR)CH₂OP(O)₂O—), the lipid-cationic polyamide conjugate isreferred to herein as a “lipitoid.” When L is a sterol moiety, (that is,a lipid moiety that is or does contain a sterol group, such as acholesterol group), the lipid-cationic polyamide conjugate is referredto herein as a “cholesteroid.” The lipid-cationic polyamide conjugate ina composition of the present invention may be a lipitoid, acholesteroid, or, in one important embodiment, a combination thereof.

In specific embodiments, R is isopropyl or 4-methoxyphenyl. A singlelipitoid or cholesteroid may include different groups R, or they may bethe same within the molecule.

The compositions of the invention result in efficient delivery of siRNAto mammalian cells effective to knockout the mRNA of a target gene.

In another aspect, a method of inhibiting expression of a target gene ina subject, which involves administering to the subject a composition asdescribed above, in which one strand of the siRNA duplex has anucleotide sequence comprised in a mRNA derived from the target gene isprovided. In another embodiment a method of manufacturing a compositionas described above for use in inhibiting expression of a target gene ina subject is provided. In a specific embodiment, a strand of the siRNAduplex includes a sequence represented by SEQ ID NO: 1, disclosedherein, with two nucleotide 3′-overhangs and phosphodiester linksthroughout, may be used, and the target gene/mRNA is Akt1.

In another aspect, the invention provides a polynucleotide deliveryvehicle composed of a mixture of at least one lipitoid and onecholesteroid. The combination lipitoid/cholesteroid delivery vehicle issuitable for delivery of a variety of polynucleotides such as plasmidDNA, antisense oligonucleotides and siRNA, to cells in compositionsincorporating such polynucleotides and the combinationlipitoid/cholesteroid delivery vehicle.

Compositions and delivery vehicles in accordance with the presentinvention may be used in vitro, for example in connection with researchincluding drug discovery, development and testing activities, or in vivofor therapeutic applications (e.g., as drugs of drug components fortreating disease) in animal, including mammalian (e.g., human) subjects.For such in vivo applications, a pharmaceutically acceptable vehicle inaccordance with the present invention is used.

Definitions

Unless otherwise noted, terminology used herein should be given itsnormal meaning as understood by one of skill in the art. In order tofacilitate understanding of the present invention, a number of definedterms are used herein to designate particular elements of the presentinvention. When so used, the following meanings are intended:

The term “lipid-conjugated polyamide” is used herein to refer to acompound having both an oligomeric amide moiety and one or more lipidmoieties. The polyamide component of the lipid-conjugated polyamidecompound may, for example, be a peptoid, in which case thelipid-conjugated polyamide may be referred to as a “lipid-conjugatedpeptoid,” in particular a cationic peptoid, in which case thelipid-conjugated polyamide may be referred to as a “lipid-cationicpeptoid conjugate.”

As used herein, the term “lipid” refers to a hydrophobic or amphipathicmoiety. A lipid moiety can be conjugated directly to the oligomericamide moiety, or optionally, indirectly to the oligomeric amide moietyvia a linker moiety. The lipid component of the lipid-conjugatedpolyamide may be or contain a non-sterol or a sterol moiety.

As used herein, the term “lipitoid” refers to a lipid-conjugated peptoidof the formula:L-X—[N(CH₂CH₂NH₂)CH₂(C═O)—N(CH₂CH₂R)CH₂(C═O)—N(CH₂CH₂R)CH₂(C═O)]₃—NH₂  (VI)wherein the lipid portion, L, is a non-sterol lipid moiety. Whereverused in the specification and claims herein, it is intended that thisformula cover positional isomers or the peptoid portion thereof, apositional isomer being any repeating three-fold motif of the peptoidportion of the formula.

As used herein, the term “cholesteroid” refers to a lipid-conjugatedpeptoid of the formula:L-X—[N(CH₂CH₂NH₂)CH₂(C═O)—N(CH₂CH₂R)CH₂(C═O)N(CH₂CH₂R)CH₂(C═O)]₃—NH₂  (VI)wherein the lipid portion, L, is a sterol lipid moiety. Wherever used inthe specification and claims herein, it is intended that this formulacover positional isomers or the peptoid portion thereof, a positionalisomer being any repeating three-fold motif of the peptoid portion ofthe formula.

The terms “oligomeric” and “oligomeric amide” are used interchangeablyherein to refer to two or more monomer units that are linked together byan amide bond,i.e., —[—(NR₁—W—CO)_(n)]_(m)—.

As used herein, the term “monomer” or “monomeric” unit refers to theunit defined by the formula—(NR₁—W—CO)—.

The terms “oligomeric reactant,” “oligomer reactant,” “oligomeric amidereactant,” and “lipid reactant” refer herein to reactive species fromwhich lipid-conjugated polyamide compounds of the present invention aresynthesized.

As used herein, the term “delivery vehicle” refers to a lipid-conjugatedpolyamide compound as further described herein that complexes with andfacilitates the delivery of a polynucleotide through a cell membrane toa target site. Delivery vehicles in accordance with the presentinvention are “pharmaceutically acceptable,” which, as used herein,refers to the compatibility of the delivery vehicles with biologicalmaterials, for example, for use in pharmaceutical formulations and inother applications, either in vivo or in vitro, where they are incontact with biological materials, such as living cells.

As used herein, the term “complex” refers to a structure formed byinteraction between two or more compounds or structures. Suchinteraction can be via chemical interaction, such as, for example,covalent, ionic, or secondary bonding (e.g., hydrogen bonding), and thelike, or via physical interaction, such as, for example, encapsulation,entrapment, and the like.

In accordance with one aspect of the present invention, lipid-conjugatedpolyamide compounds may be complexed to siRNA via covalent bondingthrough an intermediately positioned sequence of amino acids that issusceptible to degradation by endogenous proteolytic enzymes. Thus, forexample, exposure of the complex to degradative enzymes results incleavage and subsequent release of the siRNA from the complex.Lipid-conjugated polyamide compounds of the present invention can alsobe complexed to siRNA via ionic or secondary bonding, or alternativelyvia encapsulation or entrapment.

The terms “polynucleotide” and “polynucleic acid” are usedinterchangeably herein to refer to DNA, RNA, and analogues thereofpeptide-nucleic acids, as well as, DNA or RNA that has non-phosphatecontaining nucleotides. SiRNA is particularly used in accordance withthe present invention. Other polynucleotides employed in the practice ofsome aspects of the present invention can be single-stranded,double-stranded, or chimeric single- or double-stranded molecules.Specific examples include plasmid DNA and antisense oligonucleotides.

As used herein, “substituted” is meant that one or more pendanthydrogen's on an organic functional group is replaced with asubstituent, preferably selected from a halide, a lower alkyl or loweralkoxy group, halomethyl, or haloethyl.

All publications mentioned herein are incorporated herein by referencefor the purpose of disclosing and describing features of the inventionfor which the publications are cited in connection with.

Compositions

In one aspects, the present invention provides compositionsincorporating small interfering ribonucleic acid (siRNA) andlipid-conjugated polyamide based delivery vehicles. These compositionsare particularly useful in the delivery of the siRNA to cells.

1. SiRNA

The compositions of the present invention incorporate siRNAs which areeffective to specifically suppress expression of a gene of interestwithout eliciting any other activity detrimental to normal cellfunction, e.g., an interferon response. Effective siRNAs are generally,but not necessarily, chemically synthesized. In specific embodiments,siRNA directed against Akt1 messenger RNA having the sequenceCAUAGUGAGGUUGCAUCUGGUG (SEQ ID No: 1) with two nucleotide 3′-overhangsand phosphodiester links throughout may be used. In one case, the twonucleotide 3′-overhangs are TT (DNA) nucleotides. In another case, thetwo nucleotide 3′-overhangs are 2′ O-methyl UU (RNA) nucleotides.

When transfected into cells, as described in Examples 1 and 3, below,these siRNA showed very effective degradation of endogenous Akt1 mRNA,resulting in a loss of activity of the corresponding Akt1 gene. Itshould be understood that the Akt1 siRNA sequences described herein aremerely representative of a myriad other possible siRNA sequences thatmay be combined with lipid-conjugated polyamide compound deliveryvehicles in compositions in accordance with the present invention. Oneof skill in the art will appreciate for the disclosure provided hereinthat other siRNA sequences may be used in the same or a similar readilyascertainable manner to achieve the same effect for the correspondingmRNA and gene.

2. Lipid-Conjugated Polyamide Compounds

The present invention provides compositions incorporatinglipid-conjugated polyamide compound-based delivery vehicles. Suitablelipid-conjugated polyamide conjugates for use in or as these deliveryvehicles are described in co-owned PCT publications WO 98/06437 and WO99/08711 (Zuckermann et al.), based on U.S. Ser. Nos. 60/023,867,60/054,743, and 09/132,808; and in co-owned PCT publication WO 01/16306and U.S. Ser. No. 09/648,254 (Innis, et al.), based on U.S. Ser. No.60/151,246; hereby incorporated by reference in their entirety and forall purposes. These lipid-conjugated polyamide conjugates have thegeneral formula:R_(a)—[(NR₁—W—CO)_(n)]_(m)—R_(c)  (I)

-   -   wherein n is an integer selected from 1 to about 48 and m is an        integer selected from about 2 to about 48,    -   wherein R₁ for each monomeric unit, —(NR₁—W—CO)—, and R_(a) are        independently selected from the group consisting of a hydrogen        atom; a hydroxy group; an amino group; a carboxyl group; a        sulfonyl group; —SH; an optionally substituted, branched or        straight chain aliphatic group having 1 to 8 carbon atoms in a        backbone structure that optionally contains nitrogen, oxygen,        sulfur, and phosphorus, wherein said aliphatic group optionally        has one or more double or triple bonds; an optionally        substituted aryl group having 3 to 12 carbon atoms in a backbone        structure that optionally contains nitrogen, oxygen, sulfur, and        phosphorus; an optionally substituted arylalkyl group having 3        to 12 carbon atoms in a backbone structure that optionally        contains nitrogen, oxygen, sulfur, and phosphorus, wherein the        alkyl group of said arylalkyl optionally has one or more double        or triple bonds; and a lipid moiety that is optionally bonded to        linker moiety,    -   wherein R₁ is not a hydrogen atom for at least one monomeric        unit,    -   wherein R_(c) is selected from a hydrogen atom; a hydroxy group;        an amino group; a hydrazine group; a sulfonyl group; —SH; an        optionally substituted, branched or straight chain aliphatic        group having 1 to 8 carbon atoms in a backbone structure that        optionally contains nitrogen, oxygen, sulfur, and phosphorus,        wherein said aliphatic group optionally has one or more double        or triple bonds; an optionally substituted aryl group having 3        to 12 carbon atoms in a backbone structure that optionally        contains nitrogen, oxygen, sulfur, and phosphorus; an optionally        substituted arylalkyl group having 3 to 12 carbon atoms in a        backbone structure that optionally contains nitrogen, oxygen,        sulfur, and phosphorus, wherein the alkyl group of said        arylalkyl optionally has one or more double or triple bonds; and        a lipid moiety that is optionally bonded to a linker moiety,    -   wherein when R₁, R_(a), or R_(c) is an aryl or arylalkyl group        having fewer than 5 carbon atoms in a backbone structure, said        backbone structure further comprises one or more oxygen and/or        nitrogen atoms,    -   wherein W for each monomeric unit is independently selected from        an optionally substituted, branched or straight chain divalent        moiety having from 1 to about 50 atoms and optionally, one or        more double or triple bonds in a backbone that contains carbon        and optionally contains nitrogen, oxygen, sulfur, and        phosphorus, wherein said optional substitution of W may be a        lipid moiety that is optionally bonded to a linker moiety,    -   wherein said lipid moiety is a hydrophobic or amphipathic moiety        selected from the group consisting of:    -   (i) optionally substituted aryl or arylalkyl moieties having        from about 14 to about 50 carbon atoms in a backbone structure        that optionally contains nitrogen, oxygen, sulfur, and        phosphorus, wherein the alkyl group of said arylalkyl optionally        has one or more double or triple bonds; and    -   (ii) optionally substituted, branched or straight chain        aliphatic moieties having from about 10 to about 50 carbon atoms        in a backbone structure that optionally contains nitrogen,        oxygen, sulfur, and phosphorus, wherein said aliphatic moieties        optionally have one or more double or triple bonds, and    -   wherein at least one of R_(a), R_(c), W for a single monomeric        unit and R₁ for a single monomeric unit comprises a lipid        moiety.

Lipid-conjugated polyamides of the present invention can be randompolymers where each R₁ and W randomly varies from monomer to monomer(i.e., where n is 1 and m is an integer from about 2 to about 48).Alternatively, the lipid-conjugated polyamides can be polymers having mnumber of n-mers (i.e., where n is greater than 1 and m is an integerfrom about 2 to about 48) that are either repeating (i.e., each n-mer isthe same) or randomly variable (i.e., the monomer composition of eachn-mer is random).

Typically, the integer n is not more than about 40, more typically notmore than about 20, and even more typically not more than about 6.Preferably, n is about 3. The integer m is typically not more than about40, more typically not more than about 25. Usually, the integer m is notmore than about 15, typically not more than about 12, and even moretypically not more than about 8.

When R₁, R_(a), and R_(c) are aliphatic, they typically contain at least2 carbon atoms in a backbone structure and more typically contain atleast about 3 carbon atoms in a backbone structure. Aryl and arylalkylR₁, R_(a), and R_(c) groups can be linear or cyclic. Aryl and arylalkylR₁, R_(a), and R_(c) having less than 5 carbon atoms in a backbonestructure, also have one or more heteroatoms in the backbone structure,such as, nitrogen and/or oxygen. Typically aryl and arylalkyl R₁, R_(a),and R_(c) have at least about 5 carbon atoms in a backbone structure.

R_(a) is typically —OH, —H, —SH, —COOH, sulfonyl, or a lipid moietyoptionally conjugated to a linker moiety R_(c) is typically —OH, —H,—SH, —NH₂, sulfonyl, hydrazine, or a lipid moiety optionally conjugatedto a linker moiety. Preferably, either R_(a) or R_(c) is a lipid moietyoptionally conjugated to a linker moiety.

R₁ can be a sidechain that is cationic, anionic, or neutral atphysiological relevant pH. Typically, physiological pH is at least about5.5 and typically at least about 6.0. More typically, physiological pHis at least about 6.5. Usually, physiological pH is less than about 8.5and typically less than about 8.0. More typically, physiological pH isless than about 7.5.

Suitable cationic sidechains include, for example, aminoalkyl (e.g.,aminoethyl, aminopropyl, aminobutyl, aminopentyl, and the like) as wellas derivatives thereof; (S)-α-methylethylenediamino and derivativesthereof; trimethylaminoethyl and derivatives thereof; guanidinoalkyl(e.g., guanidinoethyl, guanidinopropyl, guanidinobutyl, guanidinopentyl,and the like) and derivatives thereof; aminobenzyl and derivativesthereof; pyridinium and derivatives thereof; and other like cationicmoieties that are known to those of ordinary skill in the art.

Suitable neutral sidechains include, for example, (S) or(R)-α-methylbenzyl and derivatives thereof; benzyl and derivativesthereof; phenethyl and derivatives thereof; naphthylmethyl andderivatives thereof; (S) or (R)-α-methylnaphthyl and derivativesthereof; N-propylpyrrolidinone and derivatives thereof; cyclohexylmethyland derivatives thereof; furfuryl and derivatives thereof;3,4,5-trimethoxybenzyl and derivatives thereof; methoxyethyl andderivatives thereof; p-methoxyphenethyl and derivatives thereof; isoamyl(“IsoA”) and derivatives thereof; and other like neutral moieties thatare known to those of ordinary skill in the art.

Suitable anionic sidechains include, for example, carboxy methyl,carboxy ethyl, and the like, and derivatives thereof; benzoic acid andderivatives thereof; phosphates and derivatives thereof; sulfates andderivatives thereof; and other like anionic moieties that are known tothose of ordinary skill in the art.

Optionally, R₁ can be a moiety found on naturally- ornon-naturally-occurring amino acids, or R₁ can be a lipid moietyoptionally bonded to a linker moiety. As used herein, the term“naturally-occurring amino acid” refers to Ala, Cys, Asp, Glu, Phe, His,Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr). Theterm “non-naturally-occurring amino acid” refers to amino acidstypically not found in nature, including, for example, D-isomers ofnaturally-occurring amino acids.

Typically R₁ is not hydrogen for at least two monomeric units, moretypically R₁ is not hydrogen for at least three monomeric units if n×mis 3 or more. Typically, less than about 75% of the monomer units havean R₁ that is hydrogen. More typically, less than about 50% of themonomer units have an R₁ that is hydrogen. Even more typically, lessthan about 25% of the monomer units have an R₁ that is hydrogen. Evenmore typically R₁ is not hydrogen for any of the monomeric units.

W is typically —CH₂CH₂—, —CH₂—C₆H₄—C(═O)O— (i.e., toluic acid),—CH₂CH₂—O—, —CH₂—CH═CH—, or—CR₂R₃—,  (II)

-   -   where R₂ and R₃ for each monomeric unit is independently        selected from a hydrogen atom; a hydroxy group; an amino group;        a carboxyl group; a sulfonyl group; —SH; an optionally        substituted, branched or straight chain aliphatic group having 1        to 8 carbon atoms in a backbone structure that optionally        contains nitrogen, oxygen, sulfur, phosphorus, and the like,        wherein said aliphatic group optionally has one or more double        or triple bonds; an optionally substituted aryl group having 3        to 12 carbon atoms in a backbone structure that optionally        contains nitrogen, oxygen, sulfur, phosphorus, and the like; an        optionally substituted arylalkyl group having 3 to 12 carbon        atoms in a backbone structure that optionally contains nitrogen,        oxygen, sulfur, phosphorus, and the like, wherein the alkyl        group of said arylalkyl optionally has one or more double or        triple bonds; and a lipid moiety that is optionally bonded to a        linker moiety,    -   wherein when either R₂ and R₃ is an aryl or arylalkyl group        having fewer than 5 carbon atoms in a backbone structure, said        backbone structure further comprises one or more oxygen and/or        nitrogen atoms.

When R₂ and R₃ are aliphatic, they typically contain at least 2 carbonatoms in a backbone structure and more typically contain at least about3 carbon atoms in a backbone structure. Aryl and arylalkyl R₂ and R₃groups can be linear or cyclic. Aryl and arylalkyl R₂ and R₃ having lessthan 5 carbon atoms in a backbone structure, also have one or moreheteroatoms in the backbone structure, such as, nitrogen and/or oxygen.Typically aryl and arylalkyl R₂ and R₃ have at least about 5 carbonatoms in a backbone structure.

R₂ and R₃ typically are moieties found on naturally-occurring andnon-naturally-occurring amino acids. Usually, at least one of R₂ and R₃is a hydrogen atom. Most typically, R₂ and R₃ are both hydrogen for allmonomeric units, such that compound (I) is a lipid-conjugated,N-substituted polyglycine compound.

The lipid moiety can be positioned at R_(a), R_(c), R₁ for one or moremonomers, or at a substitution position in W for one or more monomers.Lipid moieties can be bonded directly to a monomeric unit, or they canbe bonded indirectly to a monomeric unit via a linker moiety.

The term “linker” used herein refers to a moiety that functions tocouple the oligomeric amide and lipid moieties together in a manner suchthat the molecular distance between the two moieties is greater thanwould be if the lipid and oligomeric amide moieties were coupleddirectly to each other. Linker moieties can be relatively small, havingfrom 1 to about 20 atoms in a backbone, or alternatively polymeric.Small linker moieties are optionally substituted and typically have from1 to about 20 atoms in a backbone (e.g., carbon, nitrogen, oxygen,sulfur, phosphorus, and the like). Typically, small linker moieties haveless than about 18 atoms in a backbone, and more typically, less thanabout 15 atoms in a backbone. Usually, small linker moieties have lessthan about 10 atoms in a backbone, and optionally have less than about 5atoms in a backbone.

Linker moieties can be derived from bifunctional molecules such as, forexample, 6-aminohexanoic acid, 2-(2-(2-aminoethoxy)ethoxy)ethoxy) aceticacid, and the like) that are capable of reacting with both oligomericand lipid reactants. Linker moieties also can be derived from groupssuch as, for example, acyl and substituted-acyl groups, sulfonyl andsubstituted-sulfonyl groups, and other like reactive moieties that areemployed during chemical synthesis to facilitate conjugation of thelipid moiety to the oligomer moiety.

Polymeric linker moieties are optionally substituted (e.g., hydroxy-,carboxy-, phosphor amino-, and the like), substantially linear polymershaving a backbone that contains carbon, and optionally containsnitrogen, oxygen, sulfur, phosphorus, and the like. Polymeric linkermoieties have an average molecular weight between about 300 daltons andabout 15,000 daltons, typically less than about 10,000 daltons, moretypically less than about 5,000 daltons, and even more typically lessthan about 3000 daltons, and optionally less than about 1000 daltons.Suitable polymeric linker moieties include, for example, polyethyleneglycols, polypropylene glycols, polyvinyl alcohols,polyvinylpyrrolidones, and the like.

Lipid moieties are hydrophobic moieties or amphipathic moieties that areeither neutral (i.e., having no charge or a net charge of zero) orcharged, and either naturally or synthetically derived. Typically, thelipid moiety in lipid-conjugated polyamide compounds of the presentinvention is amphipathic.

Suitable lipid moieties include: (1) optionally, aryl or arylalkylmoieties having from about 14 to about 50 carbon atoms in a backbonestructure that optionally contains nitrogen, oxygen, sulfur, phosphorus,and the like, where the arylalkyl moiety optionally has one or moredouble or triple bonds; (2) optionally, branched or straight chainaliphatic moieties having from about 10 to about 50 carbon atoms in abackbone structure that optionally contains nitrogen, oxygen, sulfur,phosphorus, and the like, and optionally has one or more double ortriple bonds.

Typically, aryl and arylalkyl lipid moieties have at least about 16carbon atoms and more typically have at least about 20 carbon atoms, andeven more typically at least about 30 carbon atoms.

Aliphatic lipid moieties employed in compounds of the present inventiontypically have at least about 12 carbon atoms and more typically have atleast about 14 carbon atoms. Usually, the aliphatic lipid moieties haveat least about 18 carbon atoms, more usually at least about 24 carbonatoms, and even more usually at least about 30 carbon atoms.

The number of lipid moieties in lipid-conjugated polyamide compounds ofthe present invention can vary depending on the degree of hydrophobicitydesired, and will also vary with oligomer length (i.e., n×m) and size oflipid moiety. For example, when the lipid moiety has about 30 carbonatoms or less, lipid-conjugated polyamide compounds of the presentinvention typically have conjugated to it, a number of lipid moietiesthat is less than the number computed as 90% of the total number ofmonomeric groups (i.e., n×m) (i.e., if n is 3 and m is 3, then thenumber of lipid moieties conjugated to the lipid-conjugated polyamidecompound is typically less than about 8). More typically, when the lipidmoiety has about 30 carbon atoms or less, lipid-conjugated polyamidecompounds of the present invention have conjugated to it, a number oflipid moieties that is less than about 80% of the total number ofmonomeric groups, more typically less than about 75% of the total numberof monomeric groups, and even more typically less than about 60% of thetotal number of monomeric groups.

When the lipid moiety has more than about 30 carbon atoms, typically,lipid-conjugated polyamide compounds of the present invention haveconjugated to it a number of lipid moieties that is less than the numbercomputed as 50% of the total number of monomeric groups.

Suitable lipid moieties include those having one or more hydrophobictails that are optionally substituted aliphatic, straight chainmoieties, each independently having from about 8 to about 30 carbonatoms in a backbone that in addition, optionally contains nitrogen,oxygen, sulfur, phosphorus, and the like. Typically, hydrophobic tailshave at least about 10 carbon atoms in a backbone and more typicallyhave at least about 12 carbon atoms in a backbone. Hydrophobic tailsemployed in lipid-conjugated polyamide compounds of the presentinvention typically do not have more than about 26 carbon atoms in abackbone, and more typically do not have more than about 24 carbon atomsin a backbone.

Natural lipid moieties employed in the practice of the present inventioncan be derived from, for example, phospholipids, including, for example,phosphoglycerides (including both acyl phosphoglycerides (such as, forexample, phosphatidic acid, phosphatidyl ethanolamine, phosphatidylserine, phosphatidyl inositol, phosphatidyl inositol phosphate,phosphatidyl inositol bisphosphate, phosphatidyl glycerol,diphosphatidylglycerol, and the like) and ether phosphoglycerides);glycosylglycerides (such as, for example, monogalactosyl diacylglycerol,digalactosyldiacylglycerol, sulphoquinovosyldiacylglycerol,dimannosyldiacylglycerol, galactofuranosyldiacylglycerol,galactosylglucosyldiacylglycerol, galactosylglucosyldiacylglycerol,glucosylgalactosylglucosyldiacylglycerol, and the like); sphingolipids(such as, for example, sphingosines, glycosyl ceramides, gangliosides,and the like); and saturated and unsaturated sterols (such as, forexample, cholesterol, ergosterol, stigmasterol, sitosterol, and thelike); and other like natural lipids.

Suitable synthetic lipid moieties can be derived from, for example,dipalmitoyl phosphotidylethanolamine (DMPE) (Genzyme Corp., Cambridge),DMRIE-C™ (GibcoBRL, Gaithersburg, Md.),2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate(DOSPA) (Lipofectamine™, GibcoBRL, Gaithersburg, Md.),3β-[N—(N′,N′-dimetylaminoethyl)carbamoyl]cholesterol, Tfx-50 (PromegaCorp., Madison, Wis.),N,N1,N2,N3-tetramethyl-N,N1,N2,N3-tetrapalmitylsperimine (TM-TPS)(Cellfectin, GibcoBRL, Gaithersburg, Md.), dipalmitoylphosphatidylethanolaminospermine, and the like.

Suitable lipid moieties also include those derived from fatty acids andfatty alcohols having from about 8 to about 24 carbon atoms in abackbone. Typically, the fatty acids and fatty alcohols have at leastabout 10 carbon atoms in a backbone, and more typically have at leastabout 12 carbon atoms in a backbone. Usually, the fatty acids andalcohols from which lipid moieties are derived have less than about 20carbon atoms in a backbone.

Typically, R_(a) is a lipid moiety or a lipid moiety conjugated to alinker moiety. A particularly useful lipid moiety-containing R_(a) groupis the phosphatidyl alkylamino-substituted acyl moiety having theformula,R₄—CO—O—CH₂CH(O—CO—R₄)—CH₂—O—PO₃ ⁻—(CH₂)_(p)—NH₂ ⁺—CH₂—CO—,  (III)

-   -   where p is an integer selected from 2 or 3, and each R₄ is        independently selected from an alkyl or alkenyl moiety having        between about 6 and about 25 carbon atoms in a backbone.        Typically R₄ has up to about 22 carbon atoms in a backbone, more        typically, up to about 20 carbon atoms, even more typically up        to about 18 atoms. Typically, R₄ has at least about 8 carbon        atoms in a backbone, more typically at least about 10 carbon        atoms, and even more typically at least about 12 carbon atoms in        a backbone. Exemplary R₄ moieties include dodecyl, tridecyl,        tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, and        the like. Preferably p is 2.

Lipid-conjugated polyamide compounds of the present invention can beoptionally further conjugated or complexed with agents that impart, forexample, targeting capabilities, structural features, biologicalactivity, or that introduce degradations sites, and the like. Suitableagents include, for example, mono-, di-, and polysaccharides,polyethylene glycols, amino acids, peptides, polypeptides, proteins(including, for example, lipoproteins, glycoproteins, antibodies, andthe like), crosslinking agents, marker agents (such as, for example,fluoroscein, biotin, ³²P, and the like), and the like.

Those of ordinary skill in the art will appreciate that R₁, R_(c),R_(a), W, and the particular lipid moiety employed can be readily variedto optimize the physicochemical properties of the lipid-conjugatedpolyamide compound for delivery of a particular type of polynucleotide.For example, oligomeric moieties of the present invention suitable foruse in the delivery of siRNA to cells have a net positive charge and arecapable of condensing polynucleic acids so that they are more compact insize, thus facilitating their delivery to cells.

Compounds of formula (I) that are suitable for use in the delivery ofsiRNA to cells, include lipid-conjugated polyamide compounds havingrepeating n-mer units (i.e., where n is greater than 1). For example,when n is 3, the lipid-conjugated polyamide compound of formula (I) hasrepeating trimer units, i.e.,R_(a)—[(NR₁ ¹—W¹—CO)—(NR₁ ²—W²—CO)—(NR₁ ³—W³—CO)]_(m)—R_(c)  (IV)where R_(a), R_(c), m, each W and each R₁ are defined as in formula (I),and positional isomers thereof. Compounds having formula (IV) that aresuitable for use in the delivery of siRNA to cells include those whereR₁ ¹ is a cationic side chain, R₁ ² and R₁ ³ are both neutral sidechains, each W is CH₂, R_(c) is NH₂, and R_(a) is defined by formula(III).

Lipid-conjugated polyamide compounds of the present invention typicallyform concentration-dependent, ordered two- or three-dimensionalstructures in solution. Such structures include two dimensional arrays,such as, for example, a single charged layer or a lipid bilayer surface,and three-dimensional structures, such as, for example, micelles,vesicles, and liposomes. Typically, ordered structures formed fromlipid-conjugated polyamide compounds of the present invention bythemselves, typically are sufficiently small such that they do notscatter light. Micelles, vesicles, and liposomes prepared fromlipid-conjugated compounds complexed with polynucleotides typically haveaverage particle sizes that are less than about 1 μm, more typicallyless than about 500 nm, and even more typically less than about 200 nm.

In particular embodiments, a lipid-conjugated polyamide compound-baseddelivery vehicle of the present invention may include one or morelipid-cationic peptoid conjugates of the formula:L-X—[N(CH₂CH₂NH₂)CH₂(C═O)—N(CH₂CH₂R)CH₂(C═O)—N(CH₂CH₂R)CH₂(C═O)]₃—NH₂  (VI)and positional isomers where

-   -   L is selected from a non-steroidal lipid moiety comprising at        least one fatty alkyl or alkenyl chain between about 8 and 24        carbon atoms in length, and a sterol moiety;    -   each group R is independently selected from alkyl, aminoalkyl,        and aralkyl, and    -   X is selected from the group consisting of a direct bond, an        oligopeptide, a substantially linear alkyl chain from 2 to about        30 bonds in length, and a substantially linear chain from 2 to        about 30 bonds in length consisting of alkyl bonds and one or        more linkages selected from the group consisting of ester,        amide, carbonate, carbamate, disulfide, peptide, and ether.

When L is a non-sterol lipid moiety (that is, a lipid moiety that doesnot contain a sterol group, such as a phospholipid group (i.e.,ROOCCH₂CH(COOR)CH₂OP(O)₂O—), the lipid-cationic polyamide conjugate isreferred to herein as a “lipitoid.” When L is a sterol moiety, (that is,a lipid moiety that does contain a sterol group, such as a cholesterolgroup), the lipid-cationic polyamide conjugate is referred to herein asa “cholesteroid.” The lipid-cationic polyamide conjugate in acomposition of the present invention may be a lipitoid, a cholesteroid,or, in one important embodiment, a combination thereof.

In specific embodiments, R is isopropyl or 4-methoxyphenyl. A singlelipitoid or cholesteroid may include different groups R, or they may bethe same within the molecule.

These vehicles may be prepared by conventional solution or solid-phasesynthesis, such as are described in Zuckermann et al. cited above, andfurther detailed below. In one such procedure, the N-terminus of aresin-bound peptoid is acylated with a spacer such as Fmoc-aminohexanoicacid or Fmoc-β-alanine. After removal of the Fmoc group, the primaryamino group is reacted with a lipid moiety, such as cholesterolchloroformate, to form a carbamate linkage, e.g. as shown inCholesteroids 1 and 3 of FIG. 1. The product is then cleaved from theresin with trifluoroacetic acid and purified by reverse-phase HPLC. Afatty acid-derived lipid moiety, such as a phospholipid, may be used inplace of the steroid moiety, to form lipitoids as described herein andalso shown in FIG. 1.

The lipid moiety may also be linked to the polyamide, e.g., peptoid,moiety by other linkages, of any effective length, readily available tothe skilled practitioner. The linker is a chain up to about 30 bonds inlength, and more preferably up to about 15 bonds in length, though anyeffective length may be used. The chain is typically linear orsubstantially linear, although branched chains (including oligopeptides)and linkers containing intervening cyclic groups can also be used. Thelinker generally comprises alkyl (C—C) bonds and one or more functionalgroups such as ester, amide, carbonate, carbamate, disulfide, peptide orether bonds. The linker may comprise multiple functional groups, as in asuccinate ester or polyether, or it may be an oligopeptide, preferably a2- to 10-mer, and more preferably a 2- to 5-mer. The steroid or lipidmoiety and peptoid segment can also be joined by a direct bond.

In certain embodiments, the linker incorporates one or more bonds whichare susceptible to cleavage under appropriate conditions in vivo; forexample, hydrolyzable ester, carbonate, carbamate, or peptide bonds;disulfide bonds, which are cleavable in cellular compartments having asufficiently reducing environment; and peptide bonds, cleavable byendogenous peptidases. With respect to the latter, polypeptide linkershaving ten or fewer, or, in further embodiments, five or fewer peptidelinkages are contemplated, though longer linkers may also be used.

Representative structures of this class, shown in FIG. 1 are given thefollowing designations herein:

-   Lipitoid 1, or L1 DMPE(NaeNmpeNmpe)₃-   Lipitoid 2, or L2 DMPE(NaeNiaNia)₃-   Lipitoid 3, or L3 NtdNhd(NaeNmpeNmpe)₃-   Lipitoid 4, or L4 NddNol(NaeNmpeNmpe)₃-   Cholesteroid 1, or C1 Chol-β-ala-(NaeNmpeNmpe)₃-   Cholesteroid 3, or C3 Chol-β-ala-(NaeNiaNia)₃    wherein “Ntd” is N-tetradecylglycine; “Nhd” is N-hexadecylglycine;    “Ndd” is N-dodecylglycine; “Nol” is N-oleylglycine.

The peptoid monomers represented in the foregoing structures are asfollows:

In one aspect, the invention provides a biological agent deliveryvehicle composed of a mixture of at least one lipitoid and onecholesteroid. The delivery vehicle is suitable for delivery of a varietyof polynucleotides, such as plasmid DNA, antisense oligonucleotides andsiRNA, to cells, where the delivery vehicle is combined with thepolynucleotide(s) in a composition in accordance with the presentinvention.

Compositions in accordance with the invention result in efficientdelivery of the siRNA to mammalian cells effective knockout of targetgene mRNA. Compositions and delivery vehicles in accordance with thepresent invention may be used in vitro, for example in connection withresearch including drug discovery, development and testing activities,or in vivo for therapeutic applications (e.g., as drugs of drugcomponents for treating disease) in animal, including mammalian (e.g.,human) subjects. For such in vivo applications, a pharmaceuticallyacceptable vehicle in accordance with the present invention is used.

Synthesis of Lipid-Conjugated Polyamide Compounds

Lipid-conjugated polyamide compounds suitable for use in compositions ofthe present invention can be synthesized by solid-phase andsolution-phase methods. The present invention also provides a method ofsynthesizing lipid-conjugated polyamide compounds, said methodcomprising:

-   -   a) contacting    -   (1) a lipid reactant, with    -   (2) an oligomer reactant, wherein said oligomer reactant has the        general formula:        T_(a)-[(NR₁—W—CO)_(n)]_(m)-T_(c)  (V)    -   wherein n is an integer selected from 1 to about 48, and m is an        integer from about 2 to about 48,    -   wherein each T_(a) and T_(c) is independently selected from a        terminal group and a reactive moiety that is capable of further        reaction with said lipid reactant,    -   wherein R₁ for each monomeric unit, —(NR₁—W—CO)—, in said        oligomer reactant is selected from the group consisting of a        hydrogen atom; a hydroxy group; an amino group; a carboxyl        group; a sulfonyl group, —SH; an optionally substituted,        branched or straight chain aliphatic group having 1 to 8 carbon        atoms in a backbone structure that optionally contains nitrogen,        oxygen, sulfur, and phosphorus, wherein the aliphatic group        optionally has one or more double or triple bonds; an optionally        substituted aryl group having 3 to 12 carbon atoms in a backbone        structure that optionally contains nitrogen, oxygen, sulfur, and        phosphorus; an optionally substituted arylalkyl group having 3        to 12 carbon atoms in a backbone structure that optionally        contains nitrogen, oxygen, sulfur, and phosphorus, wherein the        alkyl group of said arylakyl optionally has one or more double        or triple bonds; and a reactive moiety that is capable of        further reaction with said lipid reactant,    -   wherein when R₁, R_(a), or R_(c) is an aryl or arylalkyl group        having fewer than 5 carbon atoms in a backbone structure, said        backbone structure further comprises one or more oxygen and/or        nitrogen atoms,    -   wherein R₁ is not a hydrogen atom for at least one monomeric        unit,    -   wherein W for each monomeric unit is selected from an optionally        substituted, branched or straight chain divalent moiety having        from 1 to about 50 atoms in a backbone that contains carbon, and        optionally contains nitrogen, oxygen, sulfur, and phosphorus,        and optionally one or more double or triple bonds, wherein said        optional substitution of W may be a reactive moiety that is        capable of further reaction with said lipid reactant,    -   wherein at least one of T_(a), T_(c), W for a single monomeric        unit, or R₁ for a single monomeric unit comprises a reactive        moiety that is capable of further reaction with said lipid        reactant; then    -   b) reacting said lipid reactant with said oligomer reactant to        conjugate the lipid reactant to the oligomer reactant.

The term “lipid reactant” used herein refers to a reactive specieshaving a lipid moiety that is capable of participating in a chemicalreaction, such as, for example, nucleophilic displacement, condensation,and the like. Lipid reactants having functional groups, such as, forexample, —NH₂, —COOH, —SH, —OH, —SO₂Cl, and —CHO are particularly usefulfor synthesizing lipid-conjugated compounds of the present invention.Lipid reactants suitable for use in the practice of the presentinvention include lipid reactants having any one of the lipid moietiesdescribed herein which can react with, or which can be modified to reactwith, the oligomeric reactant or a linker. Typically, lipid reactantsare primary, secondary, or tertiary amines. Specific lipid reactantssuitable for use herein are phosphatidylethanolamines.

As used herein, the term “oligomer reactant” refers to an oligomericamide that is capable of participating in a chemical reaction, such as,for example, nucleophilic displacement, condensation, and the like.Oligomer reactants typically are acylated with a leaving group that issusceptible to nucleophilic displacement by a nucleophile, such as anamine. Oligomer reactants suitable for use in the practice of thepresent invention include all of the oligomeric amide substituentsdescribed for formula (I) (i.e. —[(NR₁—W—CO)_(n)]_(m)—) herein.

As used herein, the term “reactive moiety” refers to a moiety that iscapable of participating in a reaction with the lipid reactant. Typicalreactive moieties include, for example, —NH₂, —OH, —H, —SH, —COOH, acyl(e.g., acetyl), benzoyl, sulfonyl (e.g., dansyl), amide, hydrazine(typically a T_(c) group), and derivatives thereof (includingalkyl-substituted derivatives), and the like. Typically, the reactivemoiety is an acyl moiety substituted with a leaving group that issusceptible to nucleophilic displacement by a nucleophile, such as anamine.

Exemplary terminal groups include moieties that are biologically activeagents, targeting agents (e.g., a cell receptor ligand, antibody, etc.),marker agents, amino acid residues that function, for example, as adegradation site for endogenous proteolytic enzymes, and the like. Theseterminal groups typically are not further reactive with the lipidreactant.

The oligomer reactant and lipid reactant can be optionally bonded toeach other via a linker moiety (which optionally can be derived from areactive moiety). Alternatively, the linker moiety, which is derivedfrom a molecule that is capable of reacting with both oligomeric andlipid reactants, can be optionally conjugated to either the lipid oroligomer reactant prior to reaction between lipid and oligomerreactants. Thus, the lipid reactant can be conjugated to the oligomerreactant either directly, or indirectly via the linker moiety.

The term “reacting” used herein refers to one or more chemical reactionsthat result in formation of a chemical bond between the lipid reactantand the oligomer reactant, either directly, or indirectly via the linkermoiety. Suitable reactions include, for example, condensation (e.g.,acylation, and the like) and nucleophilic displacement.

Oligomer reactants having the general formula (IV) can be prepared, forexample, via a series of nucleophilic displacement reactions accordingto the solid-phase method described by Zuckermann et al., PCT WO94/06451(published Mar. 31, 1994), incorporated herein by reference. The methodcan be performed utilizing automated peptide synthesis instrumentationto permit rapid synthesis of oligomer reactants of interest. Theseinstruments are commercially available from, for example, AppliedBiosystems.

Specifically, monomer assembly into oligomer reactants is achieved bythe sequential addition of “submonomer” units to the growing chain. Inone method of monomer assembly, each cycle of monomer addition consistsof two steps:

-   -   (1) acylation of a secondary amine bound to the solid support        with an acylating agent that has a leaving group (i.e., a group        susceptible to nucleophilic displacement by a nucleophile, such        as an amine) and a carbonyl group (e.g., a carboxyl group)        (i.e., the “acylation step”); followed by    -   (2) nucleophilic displacement of the leaving group with a        sufficient amount of a submonomer that has a primary, secondary,        or tertiary amino group to introduce a side-chain (i.e., the        “nucleophilic displacement step”).

Exemplary acylating agents include haloacetic acid, halomethyl benzoicacid, and the like. The efficiency of displacement of the leaving groupis modulated by the type of acylating agent employed. For example, whena haloacetic acid is employed, it has been observed that iodine is moreefficient at displacing the leaving group compared to chlorine. Suitablesubmonomers include alkylamines, alkenylamines, aromatic amines,alkoxyamines, semicarbazides, acyl hydrozides, and derivatives thereof,and the like.

Oligomer synthesis using the submonomer approach occurs in the carboxyto amino direction. The oligomer is elaborated until the desired length,then is terminated, for example, with a bromoacetamide group. Oneadvantage of using solid phase submonomer assembly to construct oligomerreactants of the present invention is that the need for N-α-protectedmonomers is eliminated, as only reactive side-chain functionalities needto be protected.

Typically, the oligomeric reactant is synthesized as a series ofrepeating di-, tri-, or tetra-mer units. An exemplary trimer-basedcationic oligomer has the following monomer sequence in the aminoterminal (T_(a)) to carboxy terminus (T_(c)) direction:

-   -   (1) positively charge monomer    -   (2) neutral monomer, and    -   (3) neutral monomer.        The terms “neutral monomer” and “positively charged monomer” as        used herein refer to the net charge of the monomeric unit. As        noted above, in other examples in accordance with the present        invention, other positional isomer motifs may be used, for        example neutral-positive-neutral; or neutral-neutral-positive.

Further reaction of the oligomer reactant with the lipid reactant canoccur by further acylation and/or nucleophilic displacement. Forexample, an oligomer reactant that is haloacylated (e.g., where T_(a) isa bromoacetyl group) can be reacted with an lipid reactant that is aprimary, secondary, or tertiary amine. Conjugation thus occurs bynucleophilic displacement of the bromine, to form a lipid-conjugatedpolyamide compound.

More specific details are provided in the following solid-phasesubmonomer synthesis protocol for lipid-cationic peptoid conjugates,including lipitoids and cholesteroids, in accordance with specificembodiments of the present invention:

General Experimental. Reagent grade solvents are used without furtherpurification. Bromoacetic acid may be obtained from Aldrich (99% grade)and DIC may be obtained from Cheminplex International. All reactions andwashings are performed at 35° C. unless otherwise noted. Washing of theresin refers to the addition of a wash solvent (usually DMF or DMSO) tothe resin, agitating the resin so that a uniform slurry is obtained(typically for about 20 seconds), followed by thorough draining of thesolvent from the resin. Solvents are best removed by vacuum filtrationthrough the fritted bottom of the reaction vessel until the resinappears dry (typically about 10 seconds). Resin slurries are agitatedvia bubbling argon up through the bottom of the fritted vessel. Solventsused to dissolve reagents should be degassed prior to use by sonicationunder house vacuum for 5 minutes. For wash solvents, it is veryconvenient to have dispensers containing DMF, DMSO and dichloromethaneavailable with adjustable volumes (1-5 mL).

If synthesis is halted and the resin is to be stored for a length oftime (overnight), it is recommended that the resin be rinsed well withmethylene chloride before storage. Resins that are to be stored may befurther dried under high vacuum. It is advisable to not stop a synthesisat the dimer stage because dimers can cyclize upon storage over a longperiod of time to form diketopiperazines.

Initial resin swelling and Fmoc Deprotection. A fritted reaction vesselis charged with 100 mg of Fmoc-Rink amide resin (0.50 mmol/g resin). Tothe resin is added 2 mL of DMF and this solution is agitated for 5minutes to swell the resin. A glass rod may be used to break up chunksof resin, if necessary. The DMF is then drained. The Fmoc group is thenremoved by adding 2 mL of 20% piperidine in DMF to the resin. This isagitated for 1 minute, then drained. Another 2 mL of 20% piperidine inDMF is added to the resin and agitated for 20 minutes, then drained. Theresin is then washed with DMF (5×2 mL).

Submonomer Synthesis cycle. The deblocked amine is acylated by adding tothe resin 850 μL of 1.2 M bromoacetic acid in DMF, followed by 175 μLneat N,N′-diisopropylcarbodiimide (DIC). This solution is agitated for20 minutes at 35° C., then drained. The resin is then washed with DMF(3×2 mL) and DMSO (1×2 mL).

The acylation step (step 1) is then followed by nucleophilicdisplacement with a primary amine (step 2). To the washed resin is added0.85 mL of a 2 M solution of the amine in DMSO. This solution isagitated for 30 min at 35° C. and then drained. The resin is then washedwith DMSO 3×2 mL) and DMF (1×2 mL). This completes one reaction cycle.

The acylation/displacement cycle is repeated until the desired oligomeris obtained. The submonomer synthesis reaction scheme is illustratedbelow:

Cleavage (for 50 μmol resin). After the synthesis reaction and resinwashing, the resin is washed with dichloromethane (2×2 mL) and dried invacuo for two hours. Prepare a solution of trifluoroacetic acid (95%,aqueous). The dried resin is placed in a glass scintillation vialcontaining a Teflon micro stir bar, and approximately 5 mL of 95%aqueous TFA is added. The scintillation vials are placed onto a stirringplate located in a fume hood. One stirring plate can accommodate four orfive vials at a time. This solution is stirred for 20 minutes. Thecleavage mixture is filtered for each sample through an 8 mL solid phaseextraction (SPE) column fitted with a 20 μm polyethylene frit into a 50mL polypropylene conical centrifuge tube. The cleavage time may need tobe lengthened depending upon which protecting groups are present in aparticular library. The resin is then washed with 1 mL of the 95% TFAand the filtrates are combined. The filtrate is then diluted with anequal volume of water in the centrifuge tube.

This solution is then frozen and lyophilized to dryness. By puncturingsmall holes in the caps of the vials, direct lyophilization from thepolypropylene tubes may be conducted. The dried product is then taken upin 10 mL of glacial acetic acid and again lyophilized to dryness. Thetwice-dried product is then taken up in 3 mL of 1:1 acetonitrile/waterand transferred to a tared 5 mL cryovial (preferably with a siliconeo-ring) and then lyophilized to dryness, generally producing a whitefluffy powder. The mass recovery can then be calculated and the productcan remain in the cryovial for cold storage. For the purpose ofcalculating yields, it may be assumed that the product is thetrifluoroacetate salt. Prior to the last lyophilization, HPLC and massspec samples should be prepared.

If the material is going to be used for testing in a biological assay,then DMSO is added to make a concentrated stock solution. Solutions at aconcentration of 100 μM/per compound or greater are preferred. This100×DMSO stock may be diluted 1:100 into buffer, yielding an assaysolution that contains 1% DMSO, and sample molecules at a concentrationof 1 μM per compound (a typical screening concentration). DMSO is a goodchoice for this because it dissolves peptoids very well, and whendiluted is compatible with most biological assays (at concentrations≦1%).

Oligomer characterization. Individual peptoid oligomers are analyzed byreverse-phase HPLC on C-18 columns (Vydac, 5 μm, 300 Å, 4.5×250 mm). Alinear gradient of 0-80% B in 40 min is used at a flow rate of 1 mL/min(solvent A=0.1% TFA in water, solvent B=0.1% TFA in acetonitrile). Majorpeaks are collected and submitted to electrospray MS analysis todetermine the molecular weights.

Lipitoid Synthesis (50 μmol scale). Lipitoids 1 and 2 (L1 and L2) arestandard peptoids where the final (N-terminal) residue is aphosphatidylethanolamine, a primary amine. L1 and L2 usedimyristrylphosphatidylethanolamine (DMPE) as the N-terminal lipidmoiety. In order to install this group, the N-terminus is bromacetylatedand washed using standard submonomer conditions (above). The resin isthen washed with 15% methanol/chlorobenzene (2×2 mL).

A 0.2 M solution of DMPE is then prepared as follows. DMPE (Genzyme) isdissolved in 15% methanol/chlorobenzene to a concentration of 0.2 M.Since this compound is in the zwitterionic form, the compound must beneutralized to obtain the amine free base. This is accomplished by theaddition of 0.92 equivalent of 50% aqueous KOH with rapid vortexing. Thebase addition may leave a small volume of water behind as a separatephase, so the sample should be centrifuged (tabletop centrifuge, max rpmfor 1 minute) and any aqueous phase removed. At this stage, the purityof the DMPE solution should be checked by TLC (TLC solvent: 80/20/0.5CH₂Cl₂/CH₃OH/NH₄OH, stain with ninhydrin). The product should have anR_(f) of ˜0.6.

The DMPE (2 mL) solution is then added to the resin. Due to thepotential frothiness of this solution, the reaction is mixed very gently(usually by intermittent argon bubbling or rotary shaking). The reactionis incubated at 35° C. overnight. The reaction mixture is then drainedand washed with 15% methanol/chlorobenzene (6×3 mL), DCE (2×3 mL) andDCM (1×3 mL). Cleavage is carried out under standard peptoid conditions(see above). Due to the increased lipophilicity of the lipitoids, HPLCanalysis should be done on a C4 column.

Lipitoids 3 and 4 (L3 and L4) are standard peptoids where the last 2N-terminal residues are made with hydrophobic amines. These amines aredissolved in 15% methanol/chlorobenzene at a concentration of 1 M. Thedisplacement reactions are performed with 1 mL of the solution at 50° C.for two hours, followed by washing with 15% methanol/chlorobenzene (3×2mL) and DMF (3×2 mL).

Cholesteroid Synthesis (50 μmol scale). The cholesterol moiety is addedby a two-step procedure where a beta-alanine linker is first added tothe N-terminus of the desired peptoid ((NaeNmpeNmpe)₃ for C1 and(NaeNiaNia)₃ for C3) followed by a coupling with cholesterolcholoroformate. Fmoc-β-alanine (NovaBiochem) is coupled to theN-terminus by the addition of 2.0 mL of a solution of Fmoc-β-alanine(0.4 M) and HOBt (0.4 M) in DMF, followed by the addition of 1.1equivalents of neat DIC. The reaction mixture is agitated for 1 hour,after which the reaction mixture is drained and the resin is washed withDMF (2×3 mL). The β-alanine coupling is then repeated, after which theresin is washed with DMF (3×3 mL) and DCE (1×3 mL). After Fmoc removal,as described above, the cholesterol moiety is then added by adding 2.0mL of a 0.4 M solution of cholesterol choloroformate (Aldrich) in DCE,followed by the addition of 1 equivalent of neat diisopropylethylamine(DIEA). The reaction mixture is mixed gently overnight at 35° C. Theresin is then washed with DCE (5×3 mL) and DCM (2×3 mL).

Since the carbamate formed is slightly acid-labile, care should be takenin the cleavage and handling of the compound. Cleavage is performed bythe addition of TFA/DCE 1:1 (5 mL) for 10 min. The filtrate is collectedand the resin washed with an additional 2 nL of cleavage cocktail. Thecombined filtrates are then dried rapidly in vacuo, and the oilresuspended in 1:1 acetonitrile/water. This is immediately frozen (−80°C.) and lyophilized. The sample should then be lyophilized once morefrom acetonitrile/water (1:1).

Lipitoid and Cholesteroid Purification. The lipitoids and cholesteroidsare purified by reverse-phase HPLC prior to use. C4 columns may be used.In one example, the compounds are dissolved in a small amount of 25%acetonitrile/water and purified on a 50×20 mm ID DuraGel HS C4 column(Peeke Scientific).). A linear gradient of 35-85% B in 40 min is used ata flow rate of 30 mL/min (solvent A=0.1% TFA in water, solvent B=0.1%TFA in acetonitrile). The combined product fractions are combined andlyophilized to a white powder.

Method for Making Lipid-Conjugated Polyamide Compositions

To prepare transfecting compositions, an aqueous solution of alipid-conjugated polyamide compound vehicle, such as a lipitoid orcholesteroid or mixture, is formulated with the siRNA, as described inExample 1. The components are preferably used in relative amounts suchthat there are at least 1.5 and preferably two to four, positive vehiclecharges for every siRNA negative charge. The exact ratio of siRNA tovehicle is preferably determined empirically for each cell type, but isgenerally in the range of 1.5-2 nmol vehicle/μg antisenseoligonucleotide. Cells may be transfected with compositions inaccordance with the present invention as described in Example 1. Furtherdetails relating to the use of compositions in accordance with thepresent invention is provided below.

Use of Lipid-Conjugated Polyamide Compositions

In another aspect, a method of inhibiting expression of a target gene ina cell, which involves administering to the cell a composition asdescribed above, in which one strand of the siRNA duplex has anucleotide sequence comprised in a mRNA derived from the target gene isprovided. The cell may be comprised in a “subject,” as defined herein.

The compositions of the present invention comprising lipid-conjugatedpolyamide compound(s) are capable of delivering an effective amount of apolynucleotide (e.g., siRNA) to cells. As used herein, the term“effective amount” refers to an amount of polynucleotide that issufficient to detectably induce, or participate in, a biologicalresponse, such as, for example, signal transduction, transcription,translation, lymphocyte activation, including, for example, antibodyproduction, and the like.

The relative quantities of lipid-conjugated polyamide compound topolynucleic acid (e.g., siRNA) are typically selected such that the+/−charge ratio of lipid-conjugated polyamide compound to polynucleotidein the composition is at least about 1.5 and less than about 10. Moretypically, the +/−charge ratio is less than about 8, and even moretypically is less than about 4. The charge ratio is computed accordingto the following:Charge Ratio=(n _(L) ×M _(L))/(3.03×M _(siRNA)),where n_(L) is the number of moles of lipid-conjugated polyamidecompound, M_(L)=net number of charges/mole lipid-conjugated polyamide,and where M_(siRNA)=micrograms of siRNA.

Compositions of the present invention can be in liquid or solid form,and can optionally include pharmaceutically acceptable excipients. Suchexcipients can be used as fillers, processing aids, delivery enhancersand modifiers, and the like. Suitable excipients include, for example,calcium phosphate, magnesium stearate, talc, monosaccharides,dissaccharides, polysaccharides, gelatin, cellulose, methyl cellulose,sodium carboxymethyl cellulose, dextrose, polyvinyl alcohol,polyvinylpyrrolidinone, low melting waxes, ion exchange resins, and thelike, as well as combinations of any two or more thereof. A thoroughdiscussion of pharmaceutically acceptable excipients is available in“Remington's Pharmaceutical Sciences” (Mack Pub. Co., NJ 1991).

Additional agents can be included in the compositions, such as, forexample, marker agents, nutrients, and the like. For example, when thebiologically active agent is a polynucleotide, agents that promoteendocytosis of the desired nucleic acids or aid in binding of thenucleic acids to the cell surface, or both, can be incorporated intocompositions of the present invention.

Liquid compositions of the present invention can be in the form of asolution, suspension, or emulsion with a liquid carrier. Suitable liquidcarriers include, for example, water, saline, pharmaceuticallyacceptable organic solvent(s), pharmaceutically acceptable oils or fats,mixtures thereof, and the like. The liquid carrier may contain othersuitable pharmaceutically acceptable additives, such as solubilizers,emulsifiers, nutrients, buffers, preservatives, suspending agents,thickening agents, viscosity regulators, stabilizers, and the like.Suitable organic solvents include, for example, monohydric alcohols,such as ethanol, and polyhydric alcohols, such as glycols. Suitable oilsinclude, for example, soybean oil, coconut oil, olive oil, saffloweroil, cottonseed oil, and the like. For parenteral administration, thecarrier can also be an oily ester such as ethyl oleate, isopropylmyristate, and the like.

Cells suitable for use in the practice of the present invention include,for example, mammalian cell lines available from the American TypeCulture Collection (ATCC), Chinese hamster ovary (CHO) cells, HeLacells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), human embryonic kidneycells, baby hamster kidney cells, mouse sertoli cells, canine kidneycells, buffalo rat liver cells, human lung cells, human liver cells,mouse mammary tumor cells, other mammalian (including human) cells(e.g., stem cells, particularly hemapoitic cells, lymphocytes,macrophages, dendritic cells, tumor cells and the like), and the like.

Suitable tissue for use as samples in the present invention include, forexample, tissue derived from mammals, such as, muscle, skin, brain,lung, liver, spleen, blood, bone marrow, thymus, heart, lymph, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, connective, and thelike.

Modes of administration to a sample include, for example, ex vivoadministration to samples derived from a subject and in vitroadministration to a sample. Methods for carrying out these modes ofadministration are well known to those of ordinary skill in the art. Forexample, ex vivo delivery and reimplantation of transformed cells into asubject can be achieved as described in e.g., International PublicationNo. WO 93/14778 (published Aug. 5, 1993).

As used herein, the term “subject” refers to cells, cell lines(including mammalian cells and cell lines), invertebrates, andvertebrates including birds and mammals, such as, for example, rodentsand humans. Direct administration to a subject can typically beaccomplished by injection, either subcutaneously, intraperitoneally,intravenously or intramuscularly or delivered to the interstital spaceof a tissue. The compositions can also be administered into a tumor orlesion. Other modes of administration include oral and pulmonaryadministration, suppositories, and transdermal applications, needles,and gene guns or hyposprays.

In addition to the delivery of siRNA (or other polynucleotides) tocells, lipid-conjugated polyamide compounds of the present invention canalso be used in applications, such as, for example, screeningpeptide-like compounds for biological activity, incorporation intobiosensors such that the oligomeric moiety has the capacity to bind to atarget ligand, and the like. For drug screening applications, forexample, libraries of lipid-conjugated polyamide compounds having avariety of R₁ groups can be synthesized and subsequently screened forbiological activity in accordance with the methods for synthesizing andscreening modified peptide libraries described in PCT publication WO91/19735 (published Dec. 26, 1991), incorporated herein by reference.

EXAMPLES

The following examples illustrate but are not intended in any way tolimit the invention.

Example 1 SiRNA Inhibition of Target mRNA

A. Preparation of Transfection Mixture

For each transfection mixture (which are examples of compositions inaccordance with the present invention), a lipitoid orlipitoid/cholesteroid combination delivery vehicle was prepared to aworking concentration of 0.5 mM in water and mixed to yield a uniformsolution. The siRNA was prepared to a working concentration of 20 μM inbuffer supplied with the siRNAs. In this example, the siRNAs were forAkt1 mRNA and had the sequence CAUAGUGAGGUUGCAUCUGGUG (SEQ ID No: 1)with two 2′ O-methyl UU (RNA) nucleotide 3′-overhangs and phosphodiesterlinks throughout (available from Integrated DNA Technologies). The siRNAwas diluted in OptiMEM™ (Gibco/BRL), in a microfuge tube, to 1 μM, orapproximately 15 μg oligo/ml of OptiMEM™. In a separate microfuge tube,vehicle, typically in the amount of about 3.75 mmol vehicle/100 pmolsiRNA, was diluted into the same volume of OptiMEM™ used to dilute thesiRNA. In this example, the starting concentrations of siRNA and vehicleused, this results in about 1.5 μl of vehicle per μl of siRNA used. Notethat the exact ratio of siRNA to vehicle must be determined empiricallyfor each cell type, but generally is about this amount. The dilutedsiRNA was immediately added to the diluted vehicle and mixed bypipetting up and down. The mixture was allowed to incubate at roomtemperature for 10 minutes.

B. Transfection

Cells were plated on tissue culture dishes one day in advance oftransfection, in growth media with serum, to yield a density attransfection of 60-90%. The siRNA/vehicle mixture was added to the cellsimmediately after mixing and incubation, to a final concentration of50-100 nM siRNA in half the normal growth medium volume. Cells wereincubated with the transfection mixture at 37° C., 5% CO₂ for 4-24hours. After incubation, the transfection mixture was diluted 2 foldwith normal growth medium with serum.

FIG. 2 shows loss of Akt1 expression when siRNA directed against Akt1mRNA is transfected into MDA435 breast cancer cells using transfectioncompositions prepared as described above. The effectiveness of threedifferent lipitoid (L2, L3) or lipitoid/cholesteroid combination (L1/C1)vehicles with a series of commercially available delivery vehicles(X=lipofectamine2000, available from Invitrogen; Y=siPORTamine,available from Ambion; and Z=siPORTlipid, available from Ambion), all inthe presence or absence of serum during transfection, is compared. Thecontrol was eg5 siRNA transfected into the cells at 100 nM concentrationusing the same vehicles and conditions. The eg5 siRNA wasdouble-stranded, all RNA, with additional two TT nucleotide 3′-overhangsand phosphodiester links throughout and had a sequence corresponding tothe DNA sequence AGAAACTAAATTACAACTTGTTA. Total RNA was extracted usingthe Roche High Pure RNA Isolation Kit, according to manufacturer'sprotocols.

The results (normalized; average of HPRT and GUSB; message levels forAkt1 were normalized to the average of the levels of the housekeepinggenes (HPRT and GUSB) to compensate for small variations in total RNAlevels among samples) show that the transfection agents and compositionsof the present invention are not substantially affected by the presenceof absence of serum and that they are very effective in reducingexpression of the target gene/mRNA.

Example 2 Loss of Luciferase Activity After siRNA Transfection

Transfection mixtures were prepared using siRNA against fireflyluciferase (CGUACGCGGAAUACUUCGA (SEQ ID No: 2); from Elbashir et al.,Nature, 411, 494 (2001)) and several different delivery vehicles inaccordance with the present invention as described herein (L1, L2,L1/C1, L4 1L1/3C1) substantially as described in Example 1. Luciferaseactivity was quantified with the Promega Dual-Luciferase Reporter AssaySystem according to package directions. As shown in FIG. 3, luciferaseactivity in MDA231 stably expressing luciferase was substantiallyreduced after transfection of an siRNA against luciferase. The controlwas a non-transfected cell line. This result provides further indicationof the capability of compositions in accordance with the presentinvention for the effective delivery of siRNA to cells.

Example 3 Knockout of Akt1 Message in Cells Transfected with siRNA

Table 1 shows data for an experiment undertaken to compare theeffectiveness of the knockout of Akt1 message in cells transfected withsiRNAs by a composition incorporating a combinationlipitoid/cholesteroid delivery vehicle in accordance with the presentinvention and using a commercially available transfection agent(Fugene6, available from Roche. HT1080 cells were transfected with 100nM of two different siRNAs (siRNA directed against Akt1 messenger RNAhaving the sequence CAUAGUGAGGUUGCAUCUGGUG (SEQ ID No: 1) with two TTnucleotide 3′-overhangs and phosphodiester links throughout or with two2′ O-methyl UU (RNA) nucleotide 3′-overhangs and phosphodiester linksthroughout) using a composition incorporating a combinationlipitoid/cholesteroid delivery vehicle 1L1/3C3) substantially accordingto the transfection mixture preparation and transfection proceduresdescribed in Example 1. Reduction in mRNA levels of about 69-83% wasobserved for compositions in accordance with the present invention. Theresults indicate that the compositions in accordance with the presentinvention are far more effective at Akt1 mRNA knockout than thecommercial Fugene6 agent. TABLE 1 ratio to 100 nM hu actin pp akt1 ppactin % mRNA KO 1:1 L1/C3 1:3 akt1#1 0.94 0.64 0.6795 69.3 1:1 L1/C3 1:3akt1#3 0.89 0.48 0.5406 82.6 1:5 Fugene6 akt1#1 0.40 0.66 1.6500 28.11:5 Fugene6 akt1#3 0.40 0.79 1.9628 9.0 HT1080 wt 0.80 1.97 2.4625 0.0

Although the foregoing invention has been described in some detail forpurposes of clarity of understanding, it will be apparent that certainchanges and modifications may be practiced within the scope of theappended claims. It should be noted that there are many alternative waysof implementing both the processes and compositions of the presentinvention. Accordingly, the present embodiments are to be considered asillustrative and not restrictive, and the invention is not to be limitedto the details given herein, but may be modified within the scope andequivalents of the appended claims.

The contents of each of the patents, patent applications and journalarticles cited above are hereby incorporated by reference herein and forall purposes as if fully set forth in their entireties.

1. A composition, comprising: a small interfering ribonucleic acid(siRNA) in a pharmaceutically acceptable delivery vehicle, the vehiclecomprising at least one compound of the formula:R_(a)—[(NR₁—W—CO)_(n)]_(m)—R_(c) wherein n is an integer selected from 1to about 48 and m is an integer selected from about 2 to about 48,wherein R₁ for each monomeric unit, —(NR₁—W—CO)—, and R_(a) areindependently selected from the group consisting of a hydrogen atom; ahydroxy group; an amino group; a carboxyl group; a sulfonyl group; —SH;an optionally substituted, branched or straight chain aliphatic grouphaving 1 to 8 carbon atoms in a backbone structure that optionallycontains nitrogen, oxygen, sulfur, and phosphorus, wherein saidaliphatic group optionally has one or more double or triple bonds; anoptionally substituted aryl group having 3 to 12 carbon atoms in abackbone structure that optionally contains nitrogen, oxygen, sulfur,and phosphorus; an optionally substituted arylalkyl group having 3 to 12carbon atoms in a backbone structure that optionally contains nitrogen,oxygen, sulfur, and phosphorus, wherein the alkyl group of saidarylalkyl optionally has one or more double or triple bonds; and a lipidmoiety that is optionally bonded to linker moiety, wherein R₁ is not ahydrogen atom for at least one monomeric unit, wherein R_(c) is selectedfrom a hydrogen atom; a hydroxy group; an amino group; a hydrazinegroup; a sulfonyl group; —SH; an optionally substituted, branched orstraight chain aliphatic group having 1 to 8 carbon atoms in a backbonestructure that optionally contains nitrogen, oxygen, sulfur, andphosphorus, wherein said aliphatic group optionally has one or moredouble or triple bonds; an optionally substituted aryl group having 3 to12 carbon atoms in a backbone structure that optionally containsnitrogen, oxygen, sulfur, and phosphorus; an optionally substitutedarylalkyl group having 3 to 12 carbon atoms in a backbone structure thatoptionally contains nitrogen, oxygen, sulfur, and phosphorus, whereinthe alkyl group of said arylalkyl optionally has one or more double ortriple bonds; and a lipid moiety that is optionally bonded to a linkermoiety, wherein when R₁, R_(a), or R_(c) is an aryl or arylalkyl grouphaving fewer than 5 carbon atoms in a backbone structure, said backbonestructure further comprises one or more oxygen and/or nitrogen atoms,wherein W for each monomeric unit is independently selected from anoptionally substituted, branched or straight chain divalent moietyhaving from 1 to about 50 atoms and optionally, one or more double ortriple bonds in a backbone that contains carbon and optionally containsnitrogen, oxygen, sulfur, and phosphorus, wherein said optionalsubstitution of W may be a lipid moiety that is optionally bonded to alinker moiety, wherein said lipid moiety is a hydrophobic or amphipathicmoiety selected from the group consisting of: (i) optionally substitutedaryl or arylalkyl moieties having from about 14 to about 50 carbon atomsin a backbone structure that optionally contains nitrogen, oxygen,sulfur, and phosphorus, wherein the alkyl group of said arylalkyloptionally has one or more double or triple bonds; and (ii) optionallysubstituted, branched or straight chain aliphatic moieties having fromabout 10 to about 50 carbon atoms in a backbone structure thatoptionally contains nitrogen, oxygen, sulfur, and phosphorus, whereinsaid aliphatic moieties optionally have one or more double or triplebonds, and wherein at least one of R_(a), R_(c), W for a singlemonomeric unit and R₁ for a single monomeric unit comprises a lipidmoiety.
 2. The composition of claim 1, wherein the delivery vehiclecomprises a lipid-cationic peptoid conjugate of the formula:L-X—[N(CH₂CH₂NH₂)CH₂(C═O)—N(CH₂CH₂R)CH₂(C═O)—N(CH₂CH₂R)CH₂(C═O)]₃—NH₂and positional isomers where L is selected from a non-sterol lipidmoiety comprising at least one fatty alkyl or alkenyl chain betweenabout 8 and 24 carbon atoms in length and a sterol moiety; each group Ris independently selected from alkyl, aminoalkyl, and aralkyl, and X isselected from the group consisting of a direct bond, an oligopeptide, asubstantially linear alkyl chain from 2 to about 30 bonds in length, anda substantially linear chain from 2 to about 30 bonds in lengthconsisting of alkyl bonds and one or more linkages selected from thegroup consisting of ester, amide, carbonate, carbamate, disulfide,peptide, and ether.
 3. The composition of claim 1, wherein said fattyalkyl or alkenyl chain is between about 14 and 24 carbon atoms inlength.
 4. The composition of claim 1, wherein L is a phospholipidgroup, having two fatty alkyl or alkenyl chains between about 8 and 24carbon atoms in length.
 5. The composition of claim 1, wherein L is acholesteryl group.
 6. The composition of claim 1, wherein R is isopropylor 4-methoxyphenyl.
 7. The composition of claim 1, wherein the deliveryvehicle comprises a lipid-cationic peptoid conjugate of the formula:L-(CH₂)_(n)—(C═O)—[N(CH₂CH₂NH₂)CH₂(C═O)—N(CH₂CH₂R)CH₂(C═O)—N(CH₂CH₂R)CH₂(C═O)]₃—NH₂where L is selected from (i) a phosphatidylethanolamino group, havingfatty alkyl or alkenyl chains between about 8 and 24 carbon atoms inlength, and (ii) a cholesteryl group linked to the adjacent —(CH₂)_(n)—segment by an ester, amide or carbamate linkage; n is 1-5; and R isselected from isopropyl and 4-methoxyphenyl.
 8. The composition of claim2, wherein the lipid-cationic peptoid conjugate is selected from thegroup consisting of compounds represented herein as: Lipitoid 1, or L1DMPE(NaeNmpeNmpe)₃ Lipitoid 2, or L2 DTMPE(NaeNiaNia)₃ Lipitoid 3, or L3NtdNhd(NaeNmpeNmpe)₃ Lipitoid 4, or L4 NddNol(NaeNmpeNmpe)₃ Cholesteroid1, or C1 Chol-β-ala-(NaeNmpeNmpe)₃ Cholesteroid 3, or C3Chol-β-ala-(NaeNiaNia)₃ and combinations thereof.
 9. The composition ofclaim 8, wherein the lipid-cationic peptoid conjugate is a combinationof a lipitoid and a cholesteroid.
 10. The composition of claim 9,wherein the lipitoid:cholesteroid ratio is about 5:1 to 1:5.
 11. Thecomposition of claim 10, wherein the lipitoid:cholesteroid ratio isabout 1:1 to 1:3.
 12. The composition of claim 11, wherein the lipitoidis L1 and the cholesteroid is C1.
 13. The composition of claim 11,wherein the lipitoid:cholesteroid ratio is about 1:3.
 14. Thecomposition of claim 1, wherein the vehicle:siRNA molar ratio is about3.75 nmol:100 pmol.
 15. The composition of claim 1, wherein the siRNAcomprises a 21-23 mer RNA duplex with two nucleotide 3′ overhangs andphophodiester links throughout.
 16. The composition of claim 15, whereinthe siRNA comprises the sequence represented by SEQ ID NO: 1, with twonucleotide 3′-overhangs and phosphodiester links throughout.
 17. Amethod of inhibiting expression of a target gene in a subject,comprising administering to the subject a composition as recited inclaim 1, wherein one strand of the siRNA duplex has a nucleotidesequence comprised in a mRNA derived from the target gene.
 18. Themethod of claim 17, wherein the delivery vehicle comprises alipid-cationic peptoid conjugate as recited in claim
 2. 19. The methodof claim 18, wherein the delivery vehicle is a combination of a lipitoidand a cholesteroid.
 20. The method of claim 19, wherein thelipitoid:cholesteroid ratio is about 5:1 to 1:5.
 21. The method of claim20, wherein the lipitoid:cholesteroid ratio is about 1:1 to 1:3.
 22. Themethod of claim 21, wherein the lipitoid is L1 and the cholesteroid isC1.
 23. The method of claim 17, wherein the siRNA comprises the sequencerepresented by SEQ ID NO: 1 with two nucleotide 3′-overhangs andphosphodiester links throughout.
 24. The method of claim 17, wherein thesubject is a mammalian cell or cell line.
 25. The method of claim 17,wherein the subject is a mammal.
 26. The method of claim 25, wherein thesubject is a human.
 27. A composition, comprising: a mixture of at leasttwo lipid-cationic peptoid conjugates, each of the formula:L-X—[N(CH₂CH₂NH₂)CH₂(C═O)—N(CH₂CH₂R)CH₂(C═O)—N(CH₂CH₂R)CH₂(C═O)]₃—NH₂and positional isomers, wherein, in a first of the compounds, L is anon-sterol lipid moiety comprising at least one fatty alkyl or alkenylchain between about 8 and 24 carbon atoms in length, and, in a second ofthe compounds, L is a sterol moiety; each group R is independentlyselected from alkyl, aminoalkyl, and aralkyl, and X is selected from thegroup consisting of a direct bond, an oligopeptide, a substantiallylinear alkyl chain from 2 to about 30 bonds in length, and asubstantially linear chain from 2 to about 30 bonds in length consistingof alkyl bonds and one or more linkages selected from the groupconsisting of ester, amide, carbonate, carbamate, disulfide, peptide,and ether.
 28. The composition of claim 27, wherein the ratio of thefirst to the second compounds in the composition is from about 5:1 to1:5.
 29. The composition of claim 28, wherein the ratio of the first tothe second compounds in the composition is from about 1:1 to 1:3. 30.The composition of claim 2, wherein the ratio of the first to the secondcompounds in the composition is about 1:1.
 31. The composition of claim2, wherein the ratio of the first to the second compounds in thecomposition is from about 1:3.
 32. The composition of claim 27, whereinthe lipid-cationic peptoid conjugates are independently selected fromthe group consisting of compounds represented herein as: Lipitoid 1, orL1 DMPE(NaeNmpeNmpe)₃ Lipitoid 2, or L2 DMPE(NaeNiaNia)₃ Lipitoid 3, orL3 NtdNhd(NaeNmpeNmpe)₃ Lipitoid 4, or L4 NddNol(NaeNmpeNmpe)₃Cholesteroid 1, or C1 Chol-β-ala-(NaeNmpeNmpe)₃ Cholesteroid 3, or C3Chol-β-ala-(NaeNiaNia)₃ and combinations thereof.
 33. The composition ofclaim 32, wherein the composition consists of L1 and C1.